Method of diagnosing adolescent idiopathic scoliosis

ABSTRACT

A method for diagnosing an increased risk for developing adolescent idiopathic scoliosis (AIS) in a human subject, comprising detecting the presence or absence of at least one impairment in melatonin-signaling pathway in a cell sample of the subject in the presence and in the absence of a known melatonin-signaling pathway agonist, wherein the cell sample is selected from the group consisting of blood cell sample, osteoblast cell sample, osteoclast cell sample and myoblast cell sample, and wherein the presence of the at least one impairment in the melatonin-signaling pathway indicates that the subject possesses an increased risk for developing AIS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/232,141 filed Sep. 11, 2008 now pending, which is acontinuation application of U.S. patent application Ser. No. 10/505,951filed Aug. 27, 2004 now abandoned, which is a National Entry Applicationof PCT application no. PCT/CA2003/00286, filed on Feb. 28, 2003 andpublished in English under PCT Article 21(2), which itself claimspriority on Canadian Patent Application 2,373,854, filed on Feb. 28,2002. All documents above are incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

The present invention relates to a method of diagnosing adolescentidiopathic scoliosis. More specifically, the present invention isconcerned with a neuroendocrinal method of diagnosing adolescentidiopathic scoliosis.

BACKGROUND OF THE INVENTION

The etiology of adolescent idiopathic scoliosis (AIS), a diseaseaffecting 0.2 to 6% of the population, is unclear. AIS affects mainlygirls in number and severity but in spite of several studies suggestinga genetic predisposition, the form of inheritance remains uncertain (3;4; 4-6). Several divergent perspectives have been postulated to betterdefine this etiology (reviewed in (2; 21-23)). Genetics, growth hormonesecretion, connective tissue structure, muscle structure, vestibulardysfunction, melatonin secretion, and platelet microstructure are majorareas of focus. The current opinion is that there is a defect of centralcontrol or processing by the central nervous system (CNS) that affects agrowing spine and that the spine's susceptibility to deformation variesfrom one individual to another.

CNS Hypothesis: B. Muscle Spindle Ontology and AIS

In 1999, Dubousset suggested that AIS is probably caused by aproprioception control problem, a neuromuscular disorder in relationwith the neurotransmitter involved with the bipedal condition.

Muscles spindles are skeletal muscle sensory organs that provide axialand limb position information (proprioception) to the CNS. It has beenproposed that muscle spindles act as muscle receptors involved in thedetection of movement, both passive and active.(5) Spindles consist ofencapsulated muscles fibers (intrafusal fibers) that are innervated byspecialized motor and sensory axons. Indeed, histologic andhistochemical analysis of the distribution of muscle spindles inparaspinal musculature of patients suffering from AIS show few musclespindles in the scoliotic muscle.(6) Although the mechanism involved inspindle ontogeny are poorly understood, the innervation of a subset ofdeveloping myotube (type I) by peripheral sensory afferents (group Ia)is a critical event for inducing intrafusal fiber differentiation andsubsequent spindle formation. The inactivation of Egr3, a zing-fingertranscription factor, by gene targeting generates mice exhibiting gaitataxia, increased frequency of perinatal mortality, scoliosis, restingtremors and ptosis. Egr3-deficient mice lacked muscles spindles, afinding that is consistent with their profound gait ataxia. Egr3 ishighly expressed in developing muscle spindles, but not in Ia afferentneurons or their terminals during developmental periods that coincidewith the induction of spindle morphogenesis by sensory afferent axons.This indicates that type I myotubes are dependent upon Egr3-mediatedtranscription for proper spindle development.(7-9) In addition, Rodgerset al., reported the detection of Pax7 expression, a member of the Paxfamily of transcription factor, in the capsules surrounding adult mousemuscle spindles where it may be implicated in the formation andmaintenance of neuromuscular contacts within the muscle spindlesthroughout life.(10) The recent report of Ichikawa et al.,(11) showingin muscle spindles the presence of OPN-immunoreactive spiral axonterminals suggest that OPN could behave as a molecular mechanoreceptorwithin the spindles. This aspect is further supported from the fact thatOPN-null mice, which are normal and viable, are not responding tobiomechanical stimuli.

Neuroendocrine Hypothesis

Recent experiments involving pinealectomy in chicken and more recentlyin rats maintained in a bipedal mode led to an alternate hypothesis.These surgeries produced a scoliosis (7; 8; 8-10) resembling in manyaspects the human disease and pointed to a neuroendocrine hypothesisinvolving a melatonin deficiency as the source for AIS. Treatment afterpinealectomy in both animal models with melatonin, the major hormone ofthe pineal gland, prevented the formation of scoliosis (11).

The biological relevance of melatonin in AIS remains controversialhowever since no significant decrease in circulating melatonin level hasbeen observed in a majority of studies (12; 13; 13; 14).

There is therefore a need for a useful method for diagnosing AIS andother diseases involving spinal deformities and for identifyingcompounds for treating these diseases.

SUMMARY OF THE INVENTION

The present invention demonstrates for the first time that AIS patientsexhibit a melatonin-signaling pathway impairment and that thisimpairment can be observed through various manifestations.

In particular, the present invention demonstrates for the first time adysfunction of melatonin-signaling in bone-forming, muscle-forming cellsand blood cells of AIS patients. In addition, the present inventiondemonstrates for the first time that non-functional Gi proteins normallycoupled to melatonin receptors MT1 and MT2 is related to suchdysfunction.

More specifically, in accordance with the present invention, there isprovided a method for diagnosing an increased risk for a diseasecharacterized by a dysfunctional melatonin-signaling pathway in ananimal, comprising detecting the presence or absence of at least oneimpairment in melatonin-signaling pathway in at least one of theanimal's cells, wherein the presence of at least one impairment inmelatonin-signaling pathway indicates that the animal possesses anincreased risk of developing said adolescent idiopathic scoliosis orother disease.

The method of the present invention may also be advantageously used todiagnose a particular type of disease characterized by a dysfunctionalmelatonin-signaling pathway by determining whether the results of theassay correspond to those of a previously tested animal affected by thisparticular type of disease. For instance, it would be possible todetermine with the method for diagnosing of the present invention,whether an animal is affected by AIS of group 1, 2 or 3 (as described inTable 2 below) by determining its osteoblast responsiveness to melatonintreatment. This is particularly interesting if the most effective drugfor treating or preventing AIS varies between the groups (1, 2 or 3).The method for diagnosing of the present invention therefore permits abetter selection of the drug to be used for a particular patient.

According to another embodiment of the present invention, there is alsoprovided a method of screening for a compound useful in the treatment ofa disease characterized by a dysfunctional melatonin-signaling pathway,said method comprising the steps of contacting a candidate compound withat least one cell expressing at least one melatonin-signaling pathwayimpairment in the presence of a known melatonin-signaling pathwayagonist, wherein the candidate compound is selected if saidmelatonin-signaling pathway impairment is reduced in the presence of thecandidate compound as compared to that in the absence thereof. Thismethod can be used for screening for compounds able to modulatemelatonin-signaling impairment generally. It can however also be used todetermine which compound is the most effective for modulating and inparticular reducing or counteracting the melatonin-pathway impairment incells from a specific group of patient or for a specific patient.Indeed, the most effective compound for these purposes may vary from onepatient to the next. The method of screening of the present inventionmay therefore be used to identify which compound is the most effectivein counteracting the melatonin-signaling pathway impairment in aspecific group of patients or in one patient in particular.

In a specific embodiment, said disease characterized by a dysfunctionalmelatonin-signaling pathway is adolescent idiopathic scoliosis or another disease involving spinal deformities. In another specificembodiment, said disease characterized by a dysfunctionalmelatonin-signaling pathway is adolescent idiopathic scoliosis. Inanother specific embodiment, said impairment is detected by anaccumulation of cyclic adenosine 5′-monophosphate (cAMP) in at least oneof said cells. In another specific embodiment, said accumulation ofcyclic adenosine 5′-monophosphate (cAMP) is induced by a known activatorof adenylyl cyclase, and wherein the inhibition of said accumulation bya known melatonin-signaling pathway agonist is detectably reduced in atleast one said cells as compared to that obtained in a control cell. Inanother specific embodiment, said known melatonin-signaling pathwayagonist is melatonin or an analog thereof. In another specificembodiment, said known melatonin-signaling pathway agonist is GTP or ananalog thereof. In another specific embodiment, said known activator ofadenylyl cyclase is forskolin or an analog thereof. In another specificembodiment, said impairment is detected by an absence of proliferationof in at least one of said cells in presence of a knownmelatonin-signaling pathway agonist. In another specific embodiment,said impairment is detected by a reduction of inhibition of osteoclastsresorption activity by the known melatonin-signaling pathway agonist,and wherein the candidate compound is selected if said reduction ofinhibition of osteoclasts resorption activity is inhibited in thepresence of the candidate compound as compared to that in the absencethereof. In another specific embodiment, said cells are selected fromthe group consisting of osteoblasts, osteoclasts, lymphocytes, monocytesand myoblasts. In another specific embodiment, said cells are bloodcells. In another specific embodiment, said cells are lymphocytes. Inanother specific embodiment, said impairment is detected by anaccumulation of cyclic adenosine 5′-monophosphate (cAMP) in said cell ascompared to that in a control cell. In another specific embodiment, themethod further comprises the step of articicially inducing saidaccumulation of cyclic adenosine 5′-monophosphate (cAMP) by a knownactivator of adenylyl cyclase.

According to a further embodiment of the present invention, there isalso provided a method of formulating a drug useful in the treatment ofa disease characterized by a dysfunctional melatonin-signaling pathway,said method comprising the steps of contacting a candidate compound withat least one cell expressing at least one melatonin-signaling pathwayimpairment, wherein the candidate compound is selected if saidmelatonin-signaling pathway impairment is reduced in the presence of thecandidate compound as compared to that in the absence thereof, andformulating said drug with said selected candidate compound.

The present invention discloses such compounds able to modulatemelatonin-signaling pathway impairment including melatonin, forskolinand estradiol.

According to specific embodiments of the present invention, the diseasecharacterized by a dysfunctional melatonin-signaling pathway isadolescent idiopathic scoliosis or another disease involving spinaldeformities. More specifically, the impairment may be detected by anaccumulation of cyclic adenosine 5′-monophosphate (cAMP) in a cell ofthe animal, an absence of said cells proliferation in presence of theknown melatonin-signaling pathway agonist, and a reduction of inhibitionof osteoclasts resorption activity induced by the knownmelatonin-signaling pathway agonist, wherein the candidate compound isselected if said reduction of inhibition of osteoclasts resorptionactivity is inhibited in the presence of the candidate compound ascompared to that in the absence thereof. Note that any cell from tissuestargeted by melatonin or expressing melatonin signalisation and whereinother pathway members do not mask melatonin-signaling impairments may beused in accordance with the methods of the present invention. The cellsused herein were selected in part for their accessibility. Hence, cellssuch as osteoblasts, osteoclasts, lymphocytes, monocytes and myoblastsare advantageously accessible and may conveniently be used in themethods of the present invention. Blood cells in particular areparticularly accessible and provide for a more rapid testing. Inspecific embodiment, said known melatonin-signaling pathway agonist ismelatonin, GTP or analogs thereof. Any other known melatonin-signalingpathway agonist may be used in accordance with the present invention. Ina specific embodiment, the known activator of adenylyl cyclase isforskolin.

Melatonin-Signaling Pathways

Melatonin-signaling pathways have been better characterized in thebrain, the pituitary gland and few peripheral tissues than bone or othermusculoskeletal tissues. Melatonin exerts its effects through specific,high-affinity receptors.(12-14) These melatonin receptors are coupled toguanine nucleotide-binding proteins (G proteins), and their activationleads to the inhibition of adenylyl cyclases, which are responsible forthe accumulation of cyclic adenosine 5′-monophosphate (cAMP) (15).Through molecular cloning, three G protein-coupled melatonin receptorsubtypes have been identified in vertebrates. The ligand-bindingproperties and signaling mechanisms of these receptors are remarkablysimilar. Each receptor subtypes is coupled to inhibition of cAMPaccumulation. The MT1 (MelR1a) and MT2 (MelR1b) receptor genes arepresent in mammals and several lines of evidence demonstrated that MT1is the receptor that mediates the reproductive and circadian responsesto melatonin. The third receptor, MelR1c (two isoforms α and β) has beenonly detected in the chicken and in Xenopus. A second type of melatoninreceptor called MT3 has been first discovered based on itspharmacological properties that are quite distinct from the MT1 and MT2receptor subtypes. Recently, the human and mouse MT3 receptors have beencloned and correspond to protein encoded by the quinone reductase 2 gene(QR2).(16) The precise role of this gene in melatonin signaltransduction remains to be determined. Besides the membranous receptors,the orphan nuclear receptors RZRα and β, have been proposed to interactwith melatonin but such interaction remains elusive. Interestingly,estrogens markedly inhibit the expression and synthesis of G proteinα-subunits (Gi1-3 and Gs) in osteoblast cultures suggesting thatmelatonin-signaling may be modulated by estrogens. (17) Furthermore,estrogens are able to increase calmodulin expression, independently ofboth estrogens receptors (ERα and β).(18) This is particularlyinteresting because calmodulin and melatonin exert a mutualantagonism,(19; 20) and membrane-bound calmodulin is able to interactwith melatonin as demonstrated in Xenopus.(21) Moreover, melatonin hasthe property to destabilize the ERs DNA-binding on their cognatesequence.(22)

Fundamental Aspects of Melatonin Signal Transduction

Expression analysis revealed that melatonin up-regulates key osteoblaststerminal differentiation markers like osteocalcin (OC), osteopontin(OPN) and bone sialoprotein (BSP). This activation was alreadydetectable after only 10 min of stimulation suggesting that melatoninstimulates osteoblast differentiation in vitro through specificinteractions with one of its membranous receptors. It was thendetermined whether this transcriptional activation was mediated by MT1or MT2 receptor subtype, and demonstrated that both receptor subtypesare expressed although time-course expression analysis revealed that MT2receptor expression was predominantly detected. At the protein level,IHC experiments demonstrated the presence of both melatonin receptors atthe cell surface. In parallel, co-immunoprecipitation assays withosteoblast purified membranes demonstrated for the first time apreferential pre-coupling of Gi proteins, Gi3>Gi2, to MT2 receptors inabsence of ligand while in presence of melatonin both proteins bindingwere increased. Similar analysis with MT1 receptor revealed that onlyGi3 was pre-coupled to this receptor. No interaction was detectedbetween Gi1 proteins and both melatonin receptors.

Bone Mineral Density in Pinealectomized Chicken

Scoliotic and non-scoliotic pinealectomized chicken showed a similar andsignificant decrease in bone mineral density suggesting that bone tissueis indeed a target of melatonin action. EMG analysis performed with thesame set of chicken showed a 75% increase in muscular tone of paraspinalmuscles on both sides while a 60% asymmetrical increase of the muscularactivity was measured on the left side, which correlated with the sideof the scoliosis curve (99% left sided).

As used herein, the expression “melatonin-signaling pathway impairment”or “dysfunction” is meant to refer to any impairment in this pathwaythat characterizes cells from patients with AIS and related syndromescausing spinal deformities and includes but is not limited to: absenceof inhibition of osteoclasts resorption activity, accumulation of cAMPin an animal cell, an hypofunctionality of Gi proteins, aphosphorylation state of Gi proteins distinct from that of normal cells,an absence of proliferation of certain cells in response to melatonin, amutation in a gene encoding a member of the melatonin signaling pathway.

As used herein, the term “control cells” is used to refer to any cellnot expressing the melatonin-signaling pathway of the cell underscrutiny. It includes cells from non-scoliotic animals and cells fromanimals displaying other types of scoliosis.

As used herein, the expression “analog thereof” is meant to include anycompound displaying the same activity as that for which the compound ofreference is used. For instance, Gpp(NH)p is an analog of GTP.

In a specific embodiment, the cAMP accumulation may have beenartificially induced by a known adenylyl cyclase activator such asforskolin and inhibited by an melatonin-signaling pathway agonist suchas melatonin itself or any agonist known for inhibiting cAMPaccumulation such as GTP or Gpp(NH)p. An absence of cAMP accumulation bythese known agonists is interpreted as a melatonin-signaling impairmentof the subject cell and of the animal from which the cell was isolated.

As used herein, the methods for diagnosing AIS and related syndromescausing spinal deformities in an animal comprises detecting anymelatonin-signaling pathway impairment in at least one of the animalcells such as but not limited to lymphocytes, monocytes, osteoclastes,osteoblasts, myoblasts from the animal, and according to specificembodiments the animal is a human.

Assays to Identify Peptides of the Present Invention

Preferred methods for testing the ability of candidate compounds tomodulate (antagonize or agonize) the melatonin-signaling pathway arepresented herein. It will be understood that the invention is not solimited. Indeed, often assays well known in the art can be used in orderto identify such compounds.

It should be understood that candidate compounds to be tested accordingto the method of the present invention include non-peptides drugcandidates (small molecules) as well as peptides targeting defectiveproteins involved in the melatonin-signaling pathway impairment, oroligonucleotides such as antisens molecules targeting a defective geneinvolved in the melatonin-signaling pathway impairment.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 graphically shows the inhibitory effect of melatonin on adenylylcyclase activity in human normal osteoblasts and in AIS osteoblasts.Distribution of single data points obtained for each AIS patients andcontrol subjects (congenital scoliosis, cong; and other scoliotic type,other/s) tested at physiological dose of melatonin (10⁻⁹M) onforskolin-stimulated osteoblasts. The black bars represent the mean ofeach group;

FIG. 2 graphically shows the inhibitory effect of melatonin on adenylylcyclase activity in human normal osteoblasts and in AIS osteoblasts.Representative experiments showing the effect of increasingconcentrations of melatonin (10⁻¹¹ to 10⁻⁵M) on forskolin-stimulatedadenylyl cyclase activity in osteoblasts from control subject andpatients with AIS (group 1, 2 and 3). Data are expressed as mean±SEM;

FIG. 3 illustrates through photographs the detection of MT1 and MT2melatonin receptors in human osteoblasts from patients with AIS and fromcontrol subjects. Each panel illustrates representative IHC experimentsperformed with MT1 receptor antibodies (upper panels) and MT2 receptorantibodies (lower panels) on primary human osteoblast cultures preparedfrom patients with AIS (AIS1-2) and compared with a control subjects;

FIG. 4 graphically shows the Gpp(NH)p inhibitory effect on adenylylcyclase activity on human osteoblasts from patients with AIS and oncontrol subjects. Distribution of single data points obtained for eachAIS patients and control subjects in presence of Gpp(NH)p (10⁻⁷M) onforskolin-stimulated osteoblasts. The black bars represent the mean ofeach group;

FIG. 5 illustrates through photographs the detection of Gi3 proteinscoupled to MT2 receptors. Immunoblots were revealed with specificantibodies reacting with individual Gi with the exception of anti-Gi3antibodies, which cross-react also with human Gi1 proteins. Note thepresence of 60 kDa bands corresponding to phosphorylated Gi3 proteins.Lane 1 and 2 are from control subject (Marfan) not treated and treatedwith melatonin (10⁻⁷ M) respectively. Lanes 3-4 and 5-6 come from twodifferent AIS patients. Lanes 7-8-9 are positive control peptides forGi1, Gi2 and Gi3 respectively;

FIG. 6 graphically shows the proliferation of human normal osteoblastsand that of AIS osteoblasts through time-courses of [3H]thymidineuptake;

FIG. 7 shows the expression analysis of melatonin receptors in humanosteoblasts;

FIG. 8 shows the effect of Gpp(NH)p (10⁻⁷M) on forskolin-stimulatedadenylyl cyclase activity in osteoblasts from control subjects and inosteoblasts from patients with AIS. Distribution of single data pointsobtained from each patient with AIS and control subjects tested;

FIG. 9 shows Gi proteins coupled to MT2 melatonin receptor. Black andwhite arrows correspond to unphosphorylated and phosphorylated forms ofGi proteins respectively;

FIG. 10 shows the detection of phosphoserine residues in phosphorylatedGi proteins. The immunoblot in FIG. 9 was stripped and reprobed withantibodies recognizing antiphosphoserine residues. Numbering correspondsto cell cultures conditions: 1) untreated; 2) with melatonin; 3) withNa3VO4, a tyrosine phosphatase inhibitor; and 4) with genistein, atyrosine kinase inhibitor;

FIG. 11 graphically shows the bone mineral density in scoliotic andcontrol chicken;

FIG. 12 graphically shows the bone mineral density in scoliotic andcontrol chicken in four different plans;

FIG. 13 graphically shows EMG activity in paraspinal musculature ofpinealectomized chicken;

FIG. 14 graphically shows EMG activity in paraspinal musculature ofpinealectomized chicken in movement;

FIG. 15 graphically shows the inhibitory effect of melatonin on adenylylcyclase activity in human normal myoblasts and in AIS myoblasts;

FIG. 16 illustrates through photographs the detection of MT1 and MT2melatonin receptors in human osteoclasts from normal human subjects.Panels labeled MT1 and MT2 represent corresponding receptor subtypedetected by IHC with specific primary antibodies and distinct secondaryantibodies conjugated to different fluorochromes (red, phycoerythrin;green, FITC). The panel labeled h-OC corresponds to a human surfaceantigen specific for mature osteoclasts. Negative control has beengenerated by omission of the primary antibodies;

FIG. 17 illustrates through photographs the detection of MT1 and MT2melatonin receptors in human osteoclasts from AIS human patients. Panelslabeled MT1 and MT2 represent corresponding receptor subtype detected byIHC with specific primary antibodies and distinct secondary antibodiesconjugated to different fluorochromes (red, phycoerythrin; green, FITC).The panel labeled h-OC corresponds to a human surface antigen specificfor mature osteoclasts. Negative control has been generated by omissionof the primary antibodies;

FIG. 18 graphically shows the measurement of osteoclasts activity (pitresorption assay) on bone matrix. The inhibitory effect of melatonin onosteoclasts activity was performed using normal human osteoclastsderived from peripheral blood. Mel, melatonin; luz, luzindole, aspecific MT2 antagonist;

FIG. 19 graphically shows the effect of estrogen on themelatonin-signaling pathway impairment of patients with AIS;

FIG. 20 shows Gi proteins coupled to MT2 melatonin receptor. The cellsused were prepared from human MG-63 osteoblast culture (panel A) andosteoblast cultures from AIS patient (case 22 of Table 1, panel B); and

FIG. 21 shows Gi proteins coupled to MT2 melatonin receptor. The cellsused were prepared from osteoblast cultures from AIS patient. Panel A,case 37 of Table 1; panel B, case 29 of Table 1.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In vitro assays were performed with bone-forming and muscle-formingcells isolated from 41 patients with adolescent idiopathic scoliosis(AIS) patients and 17 control subjects demonstrating that patients withthis disease exhibit a dysfunction of the melatonin-signaling pathway intissues targeted by this hormone.

Osteoblast and myoblast cultures prepared from specimens obtainedintraoperatively during spine surgeries were used to test the ability ofmelatonin and Gpp(NH)p, a GTP analogue, to block cAMP accumulationinduced by forskolin. In parallel, melatonin receptors and Gi proteinsfunctions were evaluated by immunohistochemistry, binding assays with[¹²⁵I]-iodomelatonin and by co-immunoprecipitation experiments. The cAMPassays demonstrated that melatonin-signaling was severely impaired inosteoblasts and myoblasts isolated from AIS patients allowing theirclassification in 3 distinct groups based upon their responsiveness tomelatonin or Gpp(NH)p. Melatonin-signaling is clearly impaired inosteoblasts and myoblasts of all AIS patients and DD patients tested.Classification of AIS patients in 3 groups suggests the presence ofdistinct mutations interfering with the melatonin signal transduction.Post-translational modifications affecting Gi protein function should beconsidered as one possible mechanism.

Experimental data showed a melatonin-signaling dysfunction inosteoblasts and myoblasts isolated from 100% AIS patients tested.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

Example 1 Clinical Characteristics of AIS Patients and Control Subjectsfrom which Osteoblasts were Isolated

The clinical characteristics of the examined AIS and control subjectsare shown in Table 1.

TABLE 1 Clinical data on patients with adolescent idiopathic scoliosisand control subjects Age at Cobb Basal Induced Melatonin Case DiagnosticCurve Pattern Gender surgery Angle Heredity cAMP cAMP Group 1 AIS Llumbar M 16.33 45 Yes 0.01 12.06 3 2 AIS L thoracolumbar F 16.17 37 Yes2.01 21.77 2 3 AIS L thoracolumbar F 17.08 47 No 0.10 15.84 2 4 AIS Lthoracolumbar F 17.92 50 na 0.16 3.81 1 5 AIS R thoracic M 16.00 49 No0.10 24.52 1 6 AIS R thoracic M 15.42 48 No 0.01 3.50 2 7 AIS R thoracicM 13.25 75 No 0.01 11.90 2 8 AIS R thoracic F 14.92 54 Yes 0.10 16.36 19 AIS R thoracic F 14.92 30 Yes 0.10 19.85 1 10 AIS R thoracic F 16.6757 Yes 0.40 12.20 2 11 AIS R thoracic F 12.67 67 Yes 0.88 48.95 3 12 AISR thoracic F 17.25 53 Yes 0.26 31.71 3 13 AIS R thoracic F 15.25 53 No0.70 20.37 2 14 AIS R thoracic F 18.42 34 No 0.46 61.08 2 15 AIS Rthoracic F 13.75 61 No 0.01 14.70 2 16 AIS R thoracic F 13.00 48 No 0.1015.84 2 17 AIS R thoracic F 16.25 60 No 0.01 13.35 2 18 AIS R thoracic F14.75 67 No 0.03 3.41 2 19 AIS R thoracic F 15.08 30 No 0.40 16.78 3 20AIS R thoracic F 14.67 32 No 0.10 21.20 3 21 AIS R thoracic F 15.83 43No 0.10 20.81 3 22 AIS R thoracolumbar M 18.67 61 Yes 0.10 22.74 1 23AIS R thoracolumbar F 14.08 50 Yes 0.90 18.70 3 24 AIS R/L doublescoliosis M 17.25 46-30 Yes 0.10 4.45 1 25 AIS R/L double scoliosis M17.17 70-50 No 1.90 67.78 3 26 AIS R/L double scoliosis F 14.17 70-48Yes 0.03 51.64 2 27 AIS R/L double scoliosis F 12.75 53-55 Yes 0.19 7.252 28 AIS R/L double scoliosis F 14.75 41-50 Yes 0.10 9.18 3 29 AIS R/Ldouble scoliosis F 16.25 51-30 No 0.10 69.91 1 30 AIS R/L doublescoliosis F 18.92 29-35 No 0.28 5.39 2 31 AIS R/L double scoliosis F14.33 57-65 No 1.20 63.40 2 32 AIS R/L double scoliosis F 19.17 45-60 No0.10 11.49 2 33 AIS R/L double scoliosis F 11.75 74-56 No 0.10 11.47 234 AIS R/L double scoliosis F 11.42 57-38 No 0.10 14.64 2 35 AIS R/Ldouble scoliosis F 18.33 23-35 No 0.41 2.30 2 36 AIS R/L doublescoliosis F 14.33 90-66 No 0.36 4.84 2 37 AIS R/L double scoliosis F12.58 61-46 No 0.23 27.28 3 38 AIS R/L double scoliosis F 15.08 90-90 No0.20 26.53 3 39 AIS R/L double scoliosis F 14.33 56-53 No 0.01 17.04 340 AIS R/L double scoliosis F 13.50 48-42 No 0.48 4.94 3 41 AIS R/Ldouble scoliosis F 12.83 59-57 No 0.10 24.99 3 42 Congenital L lumbar F18.42 53 No 0.01 1.00  2* 43 Congenital R thoracic M 14.17 45 No 0.0812.26  2* 44 Congenital R thoracic M 13.08 70 No 0.95 55.36  2* 45Congenital R thoracic F 7.42 75 No 0.10 6.74  2* 46 Cancer/spine none F10.00 0 No 0.10 45.34 Control 47 Cancer/spine L thoracic F 16.33 19 No0.12 19.55 Control 48 Chiari L thoracic M 19.92 51 Yes 0.32 15.19Control 49 DMD none M 14.00 0 No 0.16 8.80 Control 50 encéphalopath R/Ldouble scoliosis M 17.67 60-30 No 0.10 18.24 Control 51 Marfan Lthoracolumbar F 19.42 38 No 0.10 8.00 Control 52 Marfan/spondylo R/Ldouble scoliosis F 12.92 0 Yes 0.09 15.51 Control 53 NF/scoliosis Rthoracolumbar F 15.75 115 No 0.10 21.48 Control 54 Noonan R thoracic F18.75 49 No 0.01 13.97 Control 55 spondylo L lumbar F 19.00 0 No 0.1015.34 Control 56 spondylo R lumbar F 16.42 0 No 0.10 36.20 Control 57spondylo R thoracolumbar M 14.50 0 No 0.10 5.95 Control 58 Traumaticcyphosis R thoracic F 17.75 40 No 0.01 9.29 Control AIS: adolescentidiopathic scoliosis; R, right; L, left; na, not available; NF,neurofibromatosis Basal and induced cAMP values are given as pmoles/1 ×10⁵ cells

TABLE 2 Clinical data associated with individual AIS groups BasalInduced Patients Age Cobb's angle Heredity cAMP cAMP Groupe 1 F 57% 15.548°-30° 4/7 (57%) 0.11 23.09 M 43% 17 Groupe 2 F 79% 14.2 54°-47° 1/14(7%)  0.20 15.15 M 21% 16 Groupe 3 F 75% 14.3 55°-52° 3/8 (38%) 0.4228.51 M 25% 16.5 Controls mean values basal cAMP: 0.12; induced cAMP:7.58

Example 2 Study Design for Assays Performed with Osteoblasts

The melatonin signal transduction pathway functionality was investigatedin osteoblasts from patients with clinically well-defined AIS (n=41) andcompared with age- and gender-matched subjects presenting or not ascoliosis (n=17) (Table 1).

Example 3 Isolation of Human Osteoblasts

Osteoblasts were obtained intraoperatively from bone fragments reducedin smaller pieces mechanically with a bone cutter in sterile conditionsand incubated at 37° C. in 5% CO² in a 100 mm culture dish in presenceof DMEM medium containing 10% FBS (certified FBS, Invitrogen,Burlington, ON, Canada) and 1% penicillin/streptomycin (Invitrogen).After a 30-day period, osteoblasts emerged from the bone pieces wereseparated at confluence from the remaining bone fragments bytrypsinization.

Example 4 Assay for Detecting Melatonin-Signaling Pathway in AisOsteoblasts-Camp Accumulation Using Melatonin as Known Agonist of theMelatonin-Signaling Pathway

Osteoblasts from patients with AIS and control subjects were seeded inquadruplet on 24-wells plate (1×10⁵ cells/well) and incubated eitherwith the vehicle alone, dimethyl sulphoxide (DMSO, Sigma™, Oakville, ON,Canada) or the diterpene forskolin (10⁻⁵M, Sigma™) to stimulate the cAMPformation. Inhibition curves of cAMP production were generated by addingmelatonin to the forskolin-containing samples in concentrations rangingfrom 10⁻¹¹M to 10⁻⁵M in a final volume of 1 ml of DMEM media with 0.5%bovine serum albumin (BSA, Sigma™). After a 30-minute incubation at 37°C., the cells were lysed and the sample centrifuged at 4° C. The cAMPcontent was determined in 200 μl aliquot of the supernatant using anenzyme immunoassay kit (Amersham-Pharmacia Biosciences, Mississauga, ON,Canada). All assays were performed in duplicate.

In osteoblasts from control subjects, melatonin produced adose-dependent inhibition of forskolin-stimulated adenylyl cyclaseactivity detectable by a reduction of cAMP levels of about 60-70% (FIG.1-2). In contrast, osteoblasts from patients with AIS showed a lack ofinhibition of forskolin-stimulated adenylyl cyclase activity bymelatonin (FIG. 1-2). The distributions of single data points obtainedwith patients with AIS, in comparison with control subjects are reportedin FIG. 1. Further analysis allowed classifying patients with AIS into 3distinct groups according to their osteoblast responsiveness tomelatonin treatment (FIG. 2). In group 1, melatonin increased cAMPaccumulation in treated osteoblasts, which contrasted with the normalinhibitory values obtained with control subjects (FIG. 2). In group 2,osteoblasts did not response to melatonin since no significantinhibition of cAMP accumulation was observed even at pharmacologicaldose (10⁻⁷M) or higher (10⁻⁵M) as illustrated by the cAMP curveinhibition (FIG. 2). Finally, the third group showed only a weakresponse toward melatonin treatment, although at physiological dose(10⁻⁹M) no significant inhibition was measured (FIG. 2). Under standardassay conditions, basal and induced cAMP levels increased from group 1to group 3 when compared to control subjects (data not shown).

In addition, 57% of the patients in the first group (1) showed thestrongest heredity link when compared with the two other groups ofpatients. The second group of AIS patients did not respond to melatonintreatment even at pharmacological doses (10⁻⁷M) and showed basal cAMPlevels slightly elevated as compared with the first group and thecontrol subjects. The third group remained resistant to melatonin,although at higher doses of melatonin, it was possible to measure somesignificant inhibitory effects on adenylyl cyclase activity. Both basaland forskolin-stimulated cAMP levels were increased in that particulargroup when compared with the other groups and the control subjects(Tables 1 and 2). Interestingly, the severity of the disease seems to becorrelated by the augmentation of both basal and induced cAMP levelssince the third AIS group is composed of the youngest mean age of femalepatients at the time of the surgery exhibiting also the highest Cobb'sangle degrees pre-op in double scoliosis (Table 2). In spite of theheterogeneity of both groups, AIS patients displayed a more significantdysfunction of melatonin-signaling over the other types of scolioticpatients. Comparison with control subjects exhibiting also a scoliosissuggested that spinal deformities observed in distinct diseases andsyndromes could share a common pathogenic mechanism interfering with themelatonin signal transduction.

Example 5 Assay for Detecting Melatonin-Signaling Pathway Impairment inAis Osteoblasts-Camp Accumulation Using GTP or Gpp(NH)p as KnownAgonists of the Melatonin-Signaling Pathway

The functionality of Gi proteins was assessed by investigating theirability to inhibit adenylyl cyclase activity in osteoblasts. To obtaininhibition curve of cAMP production the non-hydrolysable analogue ofGTP, Gpp(NH)p (guanilyl 5′-imidophosphate, Sigma™) was added to theforskolin-containing samples in concentrations ranging from 10 nM to 100μM. The cAMP content was determined as described above in similar assayswith melatonin.

In vitro assays with Gpp(NH)p reduced cAMP levels in osteoblasts fromcontrol subjects in contrast to patients with AIS tested, which showedno inhibitory effect for a majority of AIS patients. The distribution ofsingle data points obtained from each patient is reported in FIG. 8. Thevalues reported in FIG. 8 were detected after the administration of10⁻⁹M Gpp(NH)p, a GTP non-hydrolysable analogue.

The ability of the non-hydrolysable GTP analogue to inhibit adenylylcyclase activity was detected. This enzymatic activity was previouslyamplified by forskolin. The impaired capability to inhibitforskolin-stimulated adenylyl cyclase activity was also observed withthe non-hydrolysable GTP analogue, Gpp(NH)p (FIG. 8). Considering themultiplicity of GTP-binding proteins present in osteoblasts (24), it mayseem difficult to detect a defect in a particular G-protein subtype withthe use of a GTP analogue. However, not only a preferential affinity ofGpp(NH)p towards Gi proteins in the range of concentrations used hasbeen evidenced (25; 26), but also a widely different expression of eachtype of G protein is documented, indicating that Gi are commonly 10times more abundant than Gs(24).

Analysis of the data obtained with the Gpp(NH)p assays revealed againthe presence of three distinct groups although different groups of AISpatients (based upon the melatonin assays) are retrieved. This maysuggest again that at least 3 distinct genes or type of mutationscontribute to decrease or modify Gi proteins function in AIS, whichmatches well with the clinical variables associated with each AISpatients.

Example 6 Statistical Analysis

Results from the cAMP accumulation assays are given as the mean±SEM.Data were analyzed with StatView software.

Example 7 Level of Melatonin Receptors in Osteoblasts

Patients with AIS and control subjects were also investigated to excludethe possibility that melatonin signaling dysfunction observed in thatassay could be secondary to a reduced level of melatonin receptors.RT-PCR were performed with specific primers corresponding to melatoninreceptor subtypes, MT1 (panel A, FIG. 7), MT2 (panel B, FIG. 7) and OPN(panel C, FIG. 7) using 2 μg of RNA isolated every 2 h during a 24 hcycle from MC3T3-E1 cells treated (+) or not (−) with melatonin (10⁻⁷M).Panel D, FIG. 7 represents a similar analysis with osteoblast culturesobtained from one scoliotic patient (AIS female, 15 year old) and anon-scoliotic subject (NS female, 15 year old) without (c) and withmelatonin (m, 10 ⁻⁷M). PCR products were separated on a 1.5% agarose geland visualized by ethidium bromide staining. Note that both melatoninreceptor subtypes are expressed in MC3T3-E1 cells and in humanosteoblasts but in MC3T3-E1 cells, only MT1 subtype is down regulated inpresence of melatonin while MT2 subtype is the predominant form. Nosignificant difference was observed between AIS and NS subjects.Expression analysis by RT-PCR and IHC experiments indicated nosignificant variation in melatonin receptor levels although it cannot beruled-out that their function could be altered in AIS.

Example 8 Assay for Determining Mel Receptors Function and Distributionin Osteoblasts Radioligand Binding and IHC Assays

Radioligand-binding assays with 2⁻¹²⁵I-iodomelatonin and IHC assays withMT1 and MT2 specific antibodies were performed to assess whether thedysfunction of melatonin-signaling observed could be secondary to eithera reduced level of melatonin receptors or to mutations affecting theirfunction.

To determine whether or not melatonin receptor function is affected inosteoblasts from patients with AIS, total binding assays were conductedusing the radioligand 2-[¹²⁵I] iodomelatonin (500 μM, Amersham-PharmaciaBiosciences) in the absence (total binding) or presence (non-specificbinding) of melatonin (1 μM, Sigma™). All reactions were run induplicate. The data were expressed as femtomoles of receptor permilligram of protein. Protein determination was made by the method ofBradford using BioRad™ protein assay reagents (BioRad, Mississauga, ON,Canada). Receptor subtype localization and distribution were determinedin osteoblastic cells by immunohistochemistry (IHC) assays withanti-human MT1 and anti-human MT2 antibodies (kind gifts from Prof. F.Fraschini and Dr D. Angeloni, University of Milan, Italy).

Results obtained with the radioligand binding assays showed nosignificant variation in the function of melatonin receptors (data notshown). This correlated well with IHC analysis, which revealed nosignificant variation in the synthesis and distribution of bothmelatonin receptor subtypes in both patients with AIS and controlsubjects (FIG. 3).

Example 9 Assay for Determining Gi Protein Coupling to IndividualMelatonin Receptors and Phosphorylation State in Osteoblasts

Co-immunoprecipitation assays were performed with anti-MT1 and anti-MT2specific antibodies (kind gifts of Dr Debora Angeloni and Prof FrancoFraschini, University of Milan, Italy) to identify Gi proteins coupledto individual melatonin receptor in human MG-63 osteoblasts (FIG. 9).

The specific antibodies were incubated with membrane fractions purifiedfrom osteoblasts untreated and treated with melatonin (10⁻⁹M), genisteinor herbimycin (1 μM, tyrosine kinase inhibitors, Sigma™) or sodiumorthovanadate, Na₃VO₄, (1 mM, tyrosine phosphatase inhibitor, Sigma™)for at least 16 h. Presence of coupled Gi proteins in respectiveimmune-complexes were determined by SDS-PAGE and Western blot withspecific Gi antibodies (Santa Cruz Biotechnology Inc., Santa Cruz,Calif., USA) and phosphorylation status of these coupled Gi proteinswere determined using anti-phosphoserine, phosphothreonine andphosphotyrosine antibodies (Sigma™) using the same membrane afterstripping. Purified recombinant Gi proteins were used as control forantibody specificity.

These assays showed a predominant coupling of Gi3 proteins with MT2receptor in purified osteoblast membrane fractions treated or not withmelatonin (FIG. 9) Interestingly, Western blot analysis with Gi3antibodies revealed the presence of an additional higher molecularweight band corresponding to a phosphorylated form of Gi3 proteins (thepresence of 60 kDa bands corresponding to phosphorylated Gi3 proteins inFIG. 5).

Immunodetection assay with specific antibodies reacting withphosphoproteins confirmed the presence of at least one phosphoserineresidue in those higher molecular weight Gi3 proteins (FIG. 9, 10).Furthermore, similar assays with osteoblasts isolated from two AISpatients revealed a distinct phosphorylation pattern with and withoutmelatonin addition. Western blot analysis performed with respectivemembrane fractions using antibodies reacting against individual Giproteins did not reveal any significant variation in the level of thethree Gi proteins present in human osteoblast (FIG. 5).

FIG. 20 also illustrate how the phosphorylation pattern of cellsisolated from patients with a melatonin-signaling impairment differsfrom those of control cells. Identification of Gi proteins coupled toMT2 melatonin receptor. Co-immunoprecipitation assays were performedwith specific anti-MT2 antibodies using purified membrane fractionsprepared from human MG-63 osteoblast culture (panel A) and osteoblastcultures from AIS patient (case 22, panel B) treated overnight indifferent conditions: 1-5-9) untreated; 2-6-10) with melatonin; 3-7-11)with Na₃VO₄, a tyrosine phosphatase inhibitor; and 4-8-12) withgenistein, a tyrosine kinase inhibitor. Immunoblots were revealed withspecific antibodies reacting with individual Gi with the exception ofanti-Gi3 antibodies, which cross-react also with human Gi1 proteins.Lanes 1-4 with anti-Gi1; lanes 5-8 with anti-Gi2 and lanes 9-12 withanti-Gi3. Lanes 13-15 correspond to purified recombinant Gi1-3 proteinsrespectively and were used as control for antibody specificity. The 60kDa and 43 kDa bands correspond to the phosphorylated (inactive) andunphosphorylated (active) forms of Gi protein, respectively. Note thechanges in the phosphorylation patterns occurring in Gi proteins fromAIS patient, showing increased phosphorylation and distinct regulationby kinase and phosphatase inhibitors tested. The results presented inFIG. 21 were obtained as described above and relate to cells isolatedfrom other patients: human osteoblast cultures isolated from AIS patient(panel A, case 37 of Table 1; panel B, case 29 of Table 1) treatedovernight in different conditions: 1-6-11) untreated; 2-7-12) withmelatonin; 3-9-13) with Na₃VO₄, a tyrosine phosphatase inhibitor;4-10-14) with genistein, a tyrosine kinase inhibitor; and 5-11-15) withherbimycine, another tyrosine kinase inhibitor. Lanes 1-5 with anti-Gi1;lanes 6-10 with anti-Gi2 and lanes 11-115 with anti-Gi3. Lanes 16-18correspond to purified recombinant Gi1-3 proteins respectively and wereused as control for antibody specificity. Note the changes in thephosphorylation patterns occurring in Gi proteins in both AIS patient,showing a predominant coupling with phosphorylated Gi proteins.

Expression analysis by RT-PCR did not show any significant variation inGi mRNA levels encoding for the three Gi proteins present in humanosteoblast (data not shown), although it cannot be excluded that suchvariation might occur at the protein level.

The affinities of the three Gi proteins to the MT1 and MT2 receptorsenabling them to be associated and pre-coupled to these receptorsdiffer. The Gi3 has the strongest affinity to these receptors in absenceor presence of melatonin, followed by Gi2 and then Gi1. In bothconditions, only a weak interaction of Gi1 protein was detected withboth receptor subtypes. Interestingly, in absence of melatonin, 2 formsof Gi3 and Gi2 proteins were detected suggesting that one of these formscould be phosphorylated (FIG. 12). Interestingly, overnight treatment ofthe cells with melatonin or genistein (a tyrosine kinase inhibitor)completely abolished the presence of both phosphorylated forms in MT1 orMT2 immune complexes. This suggests that a tyrosine phosphorylationregulates indirectly Gi proteins functions through the activation adownstream unknown serine kinase.

It cannot be ruled-out that changes in Gi proteins affinity for GTP andGpp(NH)p could be triggered by post-translational modifications of Giproteins involving serine residues phosphorylation. Phosphorylation ofGi proteins at their N-terminus is well known to block the formation offunctional heterotrimers with Gβ and Gγ subunits preventing theinhibition of adenylyl cyclase activity either in presence of melatoninor Gpp(NH)p.

Example 10 Clinical Characteristics of AIS Patients and Control Subjectsfrom which Myoblasts were Isolated

The clinical characteristics of the examined AIS and control subjectsare shown in Table 1 (cases 33, 22 and 8 in Table 1).

Example 11 Study Design for Myoblast Assays

The melatonin signal transduction pathway functionality was investigatedin myoblasts from patients with clinically well-defined AIS (n=3, namelycases 33, 22 and 8 in Table 1) and compared with age- and gender-matchedsubjects presenting or not a scoliosis (Table 1).

Example 12 Isolation of Human Myoblasts

Myoblasts were obtained intraoperatively from normal and AIS patientsand enzymatically dispersed, incubated, separated and put into culturesaccording to methods known in the art.

Example 13 Assay for Detecting Melatonin-Signaling Pathway Impairment inAIS Myoblasts-cAMP Accumulation Using Melatonin as Known Agonist of theMelatonin-Signaling Pathway

Preliminary tests performed with skeletal myoblasts from showed theeffect of increasing concentrations of melatonin (10⁻¹¹ to 10⁻⁵M) onforskolin-stimulated adenylyl cyclase activity in myoblasts isolatedfrom AIS patients (cases 8, 22 and 23 in Table 1). These results (FIG.15) show the incapacity of melatonin to inhibit cAMP accumulationinduced by forskolin although only a treatment with a suprapharmacological dose of melatonin (10⁻⁵M) is able in 2 cases to inhibitcAMP accumulation in myoblasts.

The functionality of melatonin signaling is assessed by investigatingthe ability of Gi proteins to inhibit stimulated adenylyl cyclaseactivity in intact skeletal myoblasts. Cells prepared from patients withAIS and control subjects are seeded in quadruplet on 24-wells plate(1×10⁵ cells/well) and incubated either with dimethyl sulphoxide (DMSO,Sigma™) or forskolin (10⁻⁵M, Sigma™) to stimulate the cAMP formation. Toobtain the inhibition curve of cAMP production, melatonin is added tothe forskolin-containing samples in concentrations ranging from 10⁻¹¹Mto 10⁻⁵M in a final volume of 1 ml of DMEM media with 0.5% bovine serumalbumin (BSA). After a 30-minute incubation at 37° C., the cells arelysed and the sample centrifuged at 4° C. The cAMP content is determinedin 200 μl aliquot of the supernatant using an enzyme immunoassay kit(Amersham-Pharmacia Biosciences).

Example 14 Assay for Detecting Melatonin-Signaling Pathway Impairment inAIS Osteoblasts-cAMP Accumulation Using Gtp or Gpp(NH)p as KnownAgonists of the Melatonin-Signaling Pathway

The functionality of Gi proteins is assessed by investigating theirability to inhibit adenylyl cyclase activity in myoblasts isolated frompatients with AIS and control subjects. To obtain inhibition curve ofcAMP production, the non-hydrolysable analogue of GTP, Gpp(NH)p(guanilyl 5′-imidophosphate, Sigma™) is added to theforskolin-containing samples in concentrations ranging from 10 nM to 100μM. Protein determination is made by the method of Bradford usingBioRad™ protein assay reagents (BioRad™) with BSA as standard. Allassays are performed in duplicate.

Basal cAMP levels is obtained from untreated cells, while cells testedwith forskolin alone corresponds to the induced levels. Standard curvefor sensitivity and quantification is performed with standards providedby the manufacturer of respective assays. (23)

Example 15 Statistical Analysis for Assays with Myoblasts

Results from the cAMP accumulation assays are given as the mean±SEM. Ananalysis of variance (ANOVA), followed by Fisher's protected leastsignificant difference (PLSD) procedure for post-hoc comparison is usedto verify the significance between 2 means.

Example 16 Assay for Determining Mel Receptors Function and Distributionin Myoblasts Radioligand Binding and IHC Assays

Cellular localization and distribution of MT1 and MT2 melatoninreceptors are determined on histological sections of human skeletalmuscles obtained intraoperatively during spine surgeries and on skeletalmyoblast cultures generated in parallel from patients with AIS andcontrol subjects.

In order to determine whether or not melatonin receptors density orfunction could be affected in skeletal myoblasts from patients with AIS,total binding assays are conducted using the radioligand 2-[¹²⁵I]iodomelatonin (Amersham-Pharmacia Biosciences). This approach is alsouseful with the primary cell cultures to determine the effects ofmelatonin pre-treatment on receptor density and function ((47; 48)).Briefly, cells are washed with phosphate buffered saline (PBS), liftedin buffer, and pelleted by centrifugation. The cells are resuspended inTris (50 mM, pH 7.4) buffer and then added to tubes containing 500 μM of2-[¹²⁵I] iodomelatonin in the absence (total) or presence (non-specific)of melatonin (1 μM) in a final reaction volume of 0.26 ml. Cells arethen incubated for 1 h at room temperature and harvested by filtrationover glass filters (Millipore™) pre-soaked in 10% polyethylenimine(Sigma™) and counted in a gamma counter. All reactions are run induplicate. The data is expressed as femtomoles of receptor per milligramof protein. Protein determination is made by the method of Bradfordusing the BioRad™ protein assay reagents (BioRad™).

IHC experiments are performed with polyclonal antibodies reactingspecifically with either the MT1 or MT2 receptor subtypes (kind giftfrom Dr Debora Angeloni and Prof Franco Fraschini, University of Milan,Italy) using a confocal microscope. In order to assess whether melatoninor estrogens could modify the cellular localization and/or distributionof MT1 or MT2 subtype, IHC experiments are performed with primary cellcultures treated with a physiological dose of melatonin (10⁻⁹M) orestradiol (10⁻¹⁰M).

Negative control for IHC is generated by omitting the primary antibodyand by competition with specific blocking peptide. Positive controls areprovided for IHC experiments and binding assays using stably transfectedC2C12 myoblastic cells expressing constitutively MT1 or MT2 receptor.Specificity of each antibody has been already tested with humanosteoblasts. In binding assays with [¹²⁵I] iodomelatonin, subtraction ofnon-specific binding obtained in presence of melatonin from the totalbinding generated with the radioligand alone determines the specificbinding. However, MT1 and MT2 can bind this radioligand with almost thesame affinity. Alternatively, the addition of luzindole (10 μM), a MT2antagonist, could reveal indirectly the contribution of individualreceptors in the total binding of 2-[¹²⁵I] iodomelatonin. All assays areperformed in duplicate. Data obtained in total binding assays with theradioligand is analysed by Student's unpaired t-test. Significance isdefined as P<0.05, and data will be analysed with StatView™ andStatistica™ softwares.

It cannot be ruled-out at this stage that scoliotic patients coulddisplay a distinct distribution of melatonin receptors. However, reducedreceptor binding in situ could indicate potential interference by anunknown factor (calmodulin, estrogens etc.), that could be easilycorrelated at least for the estrogens by similar assay in vitro. Amarked reduction of 2-[¹²⁵I]iodomelatonin binding in scoliotic sectionscould be caused by either a reduction in the number of a specificreceptor subtype or a by a mutation reducing the affinity of thisreceptor. It is unlikely that the presence of serum in the in vitrobinding assay may interfere with this assay since 10% FBS should containless than 10⁻¹¹M of melatonin.

Example 17 Assay for Determining Gi Protein Coupling to IndividualMelatonin Receptors and Phosphorylation State in Myoblasts

Muscle cells are grown to confluence in 10 cm tissue culture dishes,rinsed once with ice-cold PBS, and scraped off their plastic support.After sedimentation, the cell pellet are resuspended in 2 ml of buffer A(5 mM Tris-HCl pH 7.4/2 mM EDTA/protease inhibitors cocktail) andsubsequently disrupted by sonication. Then, membranes are sedimented bycentrifugation 450×g/5 min at 4° C. and the supernatant added on the topof 9 ml 35% sucrose cushion. Membranes will be sedimented byultracentrifugation at 150,000 xg/90 min. Purified membrane fractionsediment at the bottom of the sucrose cushion. Membrane fractions areresuspended in 1 ml of buffer B (50 mM Tris-HCl pH 7.4/5 mM MgCl₂) andincubated with or without ligand (melatonin) for 1 h at 25° C. Forligand-stimulated samples, all subsequent steps are performed in thecontinued presence of ligand. Thereafter, membranes are centrifuged at18,000×g/30 min at 4° C. and washed once in 1 ml buffer C (75 mMTris-HCl pH 7.4/12 mM MgCl₂/2 mM EDTA/protease inhibitors cocktail) andthen resuspended in the same buffer containing 1% Triton X-100 (V/V),and agitated for 3 h at 4° C. Non-solubilized membrane proteins areremoved by centrifugation at 18,000×g/30 min at 4° C.Immunoprecipitation of solubilized melatonin receptors are performedwith gentle agitation overnight at 4° C. with antibodies (1:40) reactingspecifically with either human MT1 or MT2 subtypes (kind gifts of DrDebora Angeloni and Pr Franco Fraschini, Milan University, Italy),followed by a 6 h incubation at 4° C. with 50 μl of Protein-A agarosesuspension (Sigma™) to immunoprecipitate by centrifugation theindividual melatonin receptor. G proteins are dissociated from immunecomplexes by treatments with Gpp(NH)p (0.1 mM) for 1 h at 37° C. and areseparated by 12% SDS-PAGE and transferred to nitrocellulose membranes.Immunoblot are carried-out in TBST buffer containing 5% skim milk withcommercially available antibodies recognizing individual Gi proteins(Gi1-3, Santa Cruz) and reactive bands are visualized using enhancedchemiluminescence. In parallel, similar assays are performed withpurified membranes from cells treated overnight or less withphysiological doses of melatonin, estradiol or with different kinase andphosphatase inhibitors such as tyrosine kinase inhibitors, tyrosinephosphatase inhibitors and PKC specific inhibitors. Additionalimmunodetection is performed with antibodies reacting against Gzproteins, a related Gi family member.

Example 18 Assay for Detecting Melatonin-Signaling Impairment inOsteoblasts-Proliferation Assay

An assay was performed to compare the proliferation of osteoblasts ofnormal subjects with that of scoliotic patients. In FIG. 6, Panel Arepresents time-courses experiments (triplicates) of [3H]-thymidineincorporation (cpm values in abscise axis) in human osteoblastsgenerated from bone specimens isolated from normal (NS) subject(F1/female 17 years old) and scoliotic patients (AIS M2/male 18 yearsold and F1/female, 17 years old) stimulated by a physiological dose ofmelatonin (10⁻⁹M) used as known agonist of the melatonin-signalingpathway. This assay showed that normal osteoblasts growth rate increaseslinearly in response to a physiological dose of melatonin (10⁻⁹M) whilethose from scoliotic patients did not respond to melatonin.

Example 19 Assay for Detecting Melatonin-Signaling Impairment inLymphocytes-Camp Accumulation Using Melatonin as Known Agonist of theMelatonin-Signaling Pathway

Melatonin inhibition assays of cAMP accumulation assays induced byforskolin have been performed in vitro on human lymphocytes isolatedfrom control subjects using 10 ml of blood or less.Anticoagulant-treated blood was layered on the Ficoll-Paque™ solutionand centrifuged for a short period of time. Differential migrationduring centrifugation resulted in the formation of layers containingdifferent blood cells. Because of their lower density, the lymphocyteswere found at the interface between the plasma and the Ficoll-Paque™with other slowly sedimenting particles (platelet and monocytes). Thelymphocytes were then recovered from the interface and subjected to ashort washing step with a balanced salt solution to remove anyplatelets, Ficoll-Paque™ and plasma. Then, the cells were counted andused to perform the cAMP assays described in Examples above. As is knownfrom the litterature, the lymphocytes have melatonin receptors at theirsurface. Results could be obtained in 3 h or less with this particularassay. This assay is advantageously rapid as compared to assays usingosteoblasts (at least a month) because they do not require culture time.

Example 20 Assay for Detecting Melatonin-Signaling Impairment inOsteoclasts Derived from Monocytesllymphocytes

Other functional assays with melatonin using osteoclasts derived frommonocytes/lymphocytes fraction isolated from peripheral blood of AISpatients and control subjects are performed. Primary osteoclasts arederived from the peripheral blood of patients and non-scoliotic subjectsusing 10 ml of blood or less. Anticoagulant-treated blood is layered onthe Ficoll-Paque™ solution and centrifuged for a short period of time.Differential migration during centrifugation results in the formation oflayers containing different blood cells. Because of their lower density,the lymphocytes are found at the interface between the plasma and theFicoll-Paque™ with other slowly sedimenting particles (platelet andmonocytes). The lymphocytes are then recovered from the interface andsubjected to a short washing step with a balanced salt solution toremove any platelets, Ficoll-Paque™ and plasma. The cells are thencounted and seeded at high density (1×10⁶ cells per cm²) onto artificialbone or dentin matrix in α-MEM with 10% FBS and antibiotics. After a fewdays, cells that remain adherent will start to differentiate intoosteoclasts, forming large multinucleate cells after 15-20 days.Addition of melatonin (10⁻⁹M to 10⁻⁷M) inhibits osteoclasts resorptionactivity, which is visualized by the absence of resorption pit in thebone matrix (i.e. absence of holes or fewer holes on the surface).

Different approaches (RT-PCR, immunohistochemistry) have demonstratedthe presence of both melatonin receptor subtypes (MT1 and MT2) at thesurface of human osteoclasts from normal subjects and from AIS patient(see FIGS. 16 and 17).

It was also evidenced that inhibitory activity of melatonin is mediatedin osteoclasts through the MT2 receptor since the addition of luzindole,a MT2 specific antagonist prevents or reduces the inhibitory effect ofmelatonin on osteoclasts resorption activity (See FIG. 18). It isreasonably predicted that melatonin does not affect resorbing activityof osteoclasts isolated from animals with AIS or any related syndromecausing spinal deformities contrasting with the results observed innormal human osteoclasts.

Example 21 Melatonin-Signaling Pathway Modulated by Estradiol

A method of screening of the present invention was performed andidentified estrogen as one compound able to modulate themelatonin-signaling pathway impairment in AIS patients. Experiments wereperfomed showing the effect of increasing concentrations of melatonin(10⁻¹¹ to 10⁻⁵M) used as known agonist of melatonin-signaling pathway onforskolin-stimulated adenylyl cyclase activity in osteoblasts from twopatients with AIS (group 3 see Table 2) treated or not with aphysiological dose of estradiol (10⁻¹⁰M). Results illustrated in panelsA and B of FIG. 19 correspond to AIS patient numbered 13 and 29 in Table1, respectively. It is apparent from this figure that the treatment witha physiological dose of estrogen (oe) is sufficient to further block theinhibitory effect of melatonin in scoliotic patients belonging to theAIS group 3 (see table 2).

Example 22 Evaluation of Bone Mineral Density in Scoliotic and ControlChicken

Non-invasive analyses were performed with a DEXA bone densitometer(PixiMus™ II GE Lunar) and showed a significant decrease in bone mineraldensity (BMD) in both vertebrae and femur of all chicken although nodifference was observed between those exhibiting a scoliosis and thosewithout scoliosis (FIG. 11-12). Note that in our surgical conditions therate of scoliosis in pinealectomized chicken was about 50% althoughnon-scoliotic pinealectomized chicken showed a similar BMD than thecontrols (sham or intact chicken). In FIG. 11, the chickens wereexhibiting a scoliosis 7-days post-pinealectomy, while in FIG. 12, theywere exhibiting a scoliosis 21-days post-pinealectomy. Treatment withmelatonin, 3 mg/kg/day ip, increased BMD in treated animals. Treatmentwith melatonin, 3 mg/kg/day ip, increased BMD in treated animals.

Histological analysis indicated that decreased BMD occurred particularlyat the cortical bone level (not shown).

Interestingly, EMG measurement of paraspinal musculature activity usingintra-muscular electrodes revealed a 75% bi-lateral increase in musculartone in scoliotic pinealectomized chicken at rest when compared to shamor non-scoliotic pinealectomized groups (FIG. 13). EMG analysis wasperformed with implanted electrodes 21-days post-pinealectomy. EMGactivities were recorded in active chicken and compared between sham andscoliotic pinealectomized chicken. Determination of EMG activity at restin paraspinal musculature of pinealectomized chicken.

Similar EMG analysis in active chicken showed an asymmetrical activityincreased by 30% on the left side of paraspinal musculature of scolioticchicken, corresponding to the spine deformation curve (left sided in 99%of scoliotic chicken, FIG. 14). No such effect was observed withnon-scoliotic chicken.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

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1. A method for diagnosing an increased risk for developing adolescentidiopathic scoliosis (AIS) in a human subject, comprising detecting thepresence or absence of at least one impairment in melatonin-signalingpathway in a cell sample of the subject in the presence and in theabsence of a known melatonin-signaling pathway agonist, wherein the cellsample is selected from the group consisting of blood cell sample,osteoblast cell sample, osteoclast cell sample and myoblast cell sample,and wherein the presence of the at least one impairment in themelatonin-signaling pathway indicates that the subject possesses anincreased risk for developing AIS.
 2. The method of claim 1, whereinsaid cell sample is a blood cell sample selected from the groupconsisting of lymphocyte cell sample and monocyte cell sample.
 3. Themethod of claim 1, wherein said cell sample is a lymphocyte cell sample.4. The method of claim 1, wherein said cell sample is a monocyte cellsample.
 5. The method of claim 1, wherein said known melatonin-signalingpathway agonist is melatonin or an analog thereof.
 6. The method ofclaim 1, wherein said known melatonin-signaling pathway agonist ismelatonin.
 7. The method of claim 1, wherein said knownmelatonin-signaling pathway agonist is GTP or an analog thereof.
 8. Themethod of claim 1, wherein said known melatonin-signaling pathwayagonist is GTP.
 9. The method of claim 1, wherein said at least oneimpairment is detected by an accumulation of cyclic adenosine5′-monophosphate (cAMP) in said cell sample as compared to that in acontrol cell sample.
 10. The method of claim 9, wherein saidaccumulation of cAMP is induced by a known activator of adenylylcyclase.
 11. The method of claim 10, wherein said known activator ofadenylyl cyclase is forskolin.
 12. The method of claim 9, wherein saidaccumulation of cAMP is induced by a known activator of adenylylcyclase, and the impairment is detected by an absence of inhibition ofsaid accumulation in the presence of the known melatonin-signalingpathway agonist.
 13. The method of claim 12, wherein said knownactivator of adenylyl cyclase is forskolin.
 14. The method of claim 9,wherein said accumulation of cAMP is induced by a known activator ofadenylyl cyclase, and the impairment is detected by a lower inhibitionof said accumulation in the presence of the known melatonin-signalingpathway agonist in the cell sample as compared to that in a control cellsample.
 15. The method of claim 14, wherein said known activator ofadenylyl cyclase is forskolin.
 16. The method of claim 9, wherein saidaccumulation of cAMP is induced by a known activator of adenylylcyclase, and wherein inhibition of said accumulation by the knownmelatonin-signaling pathway agonist is detectably reduced in the cellsample as compared to that in a control cell sample.
 17. The method ofclaim 16, wherein said known activator of adenylyl cyclase is forskolin.18. The method of claim 1, wherein said at least one impairment isdetected by a reduction of inhibition of osteoclast resorption activityin the presence of the known melatonin-signaling pathway agonist. 19.The method of claim 1, wherein said impairment is detected by an absenceof proliferation of at least one of said cells in the presence of theknown melatonin-signaling pathway agonist.